For the sake of simplicity, Fig. 10.4 omitted one stepof cDNA library construction. The figure impliedthat the last step of the process is the ligation ofblunt-ended cDNAs into plasmid cloning vectors.Although such ligation reactions can occur, in realitythey are highly inefficient. Instead, scientists convertblunt-ended cDNA molecules into sticky-ended molecules using adapters, and then they ligate thecDNAs into vectors with compatible sticky ends.Adapters are short, partly double-stranded DNAmolecules made by hybridization of two singlestranded oligonucleotides made in a DNA synthesizer.Suppose that the following two oligonucleotides weresynthesized and then mixed together at high concentration and at a temperature that promotes hybridization of complementary DNA sequences:5′ CCCCCG 3′5′ AATTCGGGGG 3′a. Draw the hybridized DNA molecules. These arethe adapters.b. Suppose you added the adapters and ligase enzymeto blunt-ended cDNAs at a very high molar ratio ofadapters to cDNAs, so that each cDNA molecule isligated to one adapter at each of its ends. Draw apicture of a resulting cDNA molecule.
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
For the sake of simplicity, Fig. 10.4 omitted one step
of cDNA library construction. The figure implied
that the last step of the process is the ligation of
blunt-ended cDNAs into plasmid cloning vectors.
Although such ligation reactions can occur, in reality
they are highly inefficient. Instead, scientists convert
blunt-ended cDNA molecules into sticky-ended molecules using adapters, and then they ligate the
cDNAs into vectors with compatible sticky ends.
Adapters are short, partly double-stranded DNA
molecules made by hybridization of two singlestranded oligonucleotides made in a DNA synthesizer.
Suppose that the following two oligonucleotides were
synthesized and then mixed together at high concentration and at a temperature that promotes hybridization of complementary DNA sequences:
5′ CCCCCG 3′
5′ AATTCGGGGG 3′
a. Draw the hybridized DNA molecules. These are
the adapters.
b. Suppose you added the adapters and ligase enzyme
to blunt-ended cDNAs at a very high molar ratio of
adapters to cDNAs, so that each cDNA molecule is
ligated to one adapter at each of its ends. Draw a
picture of a resulting cDNA molecule.
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